Fig. 5: Correlation between mutational landscape and tumour ecology.

a–c Quantification of immune infiltrate (CD45⁺ cells/mm²), T-cell infiltrate (CD3⁺ cells/mm²) and macrophage infiltrate (CD68⁺ cells/mm²) in the different tumour grades (hyperplasia n = 18, mild dysplasia n = 12, moderate/severe dysplasia n = 16, invasive SCC n = 12 mice, 2 sections/mouse and >6 fields quantified per section; means ± SD are shown, non-significant, one-way ANOVA). d Quantification of proliferation (EdU⁺ cells/mm² in the different tumour grades (hyperplasia n = 15, mild dysplasia n = 12, moderate/severe dysplasia n = 14, invasive SCC n = 11, 2 sections/mouse and >6 fields quantified per section; means ± SD are shown. Group comparison with one-way ANOVA, non-significant. Multiple comparison: unpaired t-tests between each grade, non-significant for all except between invasive SCC and hyperplasia, *p < 0.05, p = 0.0262). e Linear regression analysis to establish the relationship between the total number of mutations in each gene (columns) and the clinical parameters (rows). The heatmap shows the p-value for each correlation (Supplementary Data 7). f Comparison of immune infiltrate (CD45⁺/mm²) between Notch1 mutated and Notch1 non-mutated (wt) tumours (n = 56 Notch1 wild-type lesions and n = 6 Notch1 mutated lesions. Bar graphs represent mean ± SD, *p < 0.05, p = 0.0083, unpaired one-tailed t-test). g, h Quantification of immune infiltrate of CD45⁺ cells (g) and T-cell infiltrate (CD3⁺ cells) (h) in Notch1 mutated tumours. Immunofluorescence staining of representative sections of Notch1 wt and Notch1 mutated tumours labelled with anti-krt14 (green) and anti-CD45 or anti-CD3 (red), counterstained with nuclear dye DAPI. Immune infiltrate was quantified by the number of CD45⁺ or CD3⁺ cells per mm² (Supplementary Fig. 4a, b). All tumours were analysed and for each tumour ≥6 regions were quantified per section (n = 2–3 sections/tumour/staining). g idx2, CD45⁺ immune infiltrate index 2 (2000–5000 cells/mm²); idx3, index 3 (5000–10,000 cells/mm²); idx4, index 4 (>10,000 cells/mm²). h idx1, CD3⁺ immune infiltrate index 1 (<500cells/mm²); idx2, index 2 (500–1000 cells/mm²); idx3, index 3 (>1000 cells/mm²). Scale bars 100 µm, 20 µm in magnified squares.